The proposed research program reflects the research of the Principal Investigator, Michael P. Pollastri and his collaborator Dr. Robert Campbell at the Marine Biological Laboratory at Woods Hole in development of new compounds that hold promise as therapeutics for neglected diseases. In particular, since the genome for Trypanosoma brucei (the causative parasite for Human African Trypanosomiasis (HAT)) and T. cruzi (which causes Chagas disease) have been recently elucidated, a number of potentially therapeutically-relevant molecular targets have been identified. Moreover, several of these molecular targets display a moderate-to- high level of homology to orthologous human targets that have been well-explored in the biopharmaceutical industry for the treatment of human disease. The overarching goal of this project is to develop compounds that display a high level of inhibition of two trypanosomal phosphodiesterases, TbrPDEB1 and TbrPDEB2, enzymes that have been previously demonstrated by validation experiments (RNAi) to be required for parasite virulence. These parasitic enzymes show ~30-35% homology to, and the parasitic enzyme active sites are highly similar to those of the human enzymes. This project will seek to (1) purify and biochemically characterize the parasitic enzymes TbrPDEB1 and TbrPDEB2 to establish cell-free assays suitable for testing compounds for inhibitory activity during hit and lead optimization, (2) identify the best class of existing hPDE inhibitors for pursuit by medicinal chemistry optimization, and (3) optimize potent, selective inhibitor compounds that have in vivo activity (tested in collaboration with the World Health Organization) for the oral dosing and blood brain barrier penetration essential for activity in the most severe forms of HAT. This project is intended to deliver compounds suitable for consideration as clinical candidates. Recognizing that the optimization of compounds from screening for this purpose is a major gap in drug discovery for neglected diseases the investigators have designed the project to i) focus on meeting the clear Target Product Profile established by WHO for HAT and ii) promote and involve collaboration from experts from industry drug discovery, recombinant enzyme production and characterization, medicinal chemistry, ADME optimization, and the assessment of compounds against hit and lead criteria in well-established in vitro and in vivo models of the primary indication HAT. The investigators will also utilize data sharing practices already established in the community of researchers working on other neglected disease targets, so that the work can contribute to additional efforts against HAT and other illnesses that compromise the overall quality of global health. Human African trypanosomiasis (HAT), also known as Sleeping Sickness, is a serious affliction that affects up to 300,000-500,000 people per year in sub-Saharan Africa, leading to approximately 60,000 deaths annually. A parasitic disease transmitted by the bite of an inflected tsetse fly, HAT is fatal if untreated, yet current drug therapies are not optimal from the perspective of toxicity, dosage regimens, or cost. All of these limitations contribute to lower rates of patient compliance, reducing the likelihood of successful treatments. This project will validate and develop a class of compounds that inhibit two key phosphodiesterase enzymes that have been identified in Trypanosoma sp. and been implicated in parasite viability. Guided by previous results from drug discovery programs that have targeted the human version of these enzymes, we will develop new compounds that target these parasitic enzymes in order to (1) confirm that their inhibition by small molecule drugs leads to parasite death, and (2) produce new drug substances for advancement into clinical studies for this disease.